Transcription factor Fra-1 targets arginase-1 to enhance macrophage-mediated inflammation in arthritis

Abstract

The polarization of macrophages is regulated by transcription factors such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1). In this manuscript, we delineated the role of the transcription factor Fos-related antigen 1 (Fra-1) during macrophage activation and development of arthritis. Network level interaction analysis of microarray data derived from Fra-1- or Fra-2-deficient macrophages revealed a central role of Fra-1, but not of Fra-2 in orchestrating the expression of genes related to wound response, toll-like receptor activation and interleukin signaling. Chromatin-immunoprecipitation (ChIP)-sequencing and standard ChIP analyses of macrophages identified arginase 1 (Arg1) as a target of Fra-1. Luciferase reporter assays revealed that Fra-1 down-regulated Arg1 expression by direct binding to the promoter region. Using macrophage-specific Fra-1- or Fra-2- deficient mice, we observed an enhanced expression and activity of Arg1 and a reduction of arthritis in the absence of Fra-1, but not of Fra-2. This phenotype was reversed by treatment with the arginase inhibitor Nω-hydroxy-nor-L-arginine, while ʟ-arginine supplementation increased arginase activity and alleviated arthritis, supporting the notion that reduced arthritis in macrophage-specific Fra-1-deficient mice resulted from enhanced Arg1 expression and activity. Moreover, patients with active RA showed increased Fra-1 expression in the peripheral blood and elevated Fra-1 protein in synovial macrophages compared to RA patients in remission. In addition, the Fra-1/ARG1 ratio in synovial macrophages was related to RA disease activity. In conclusion, these data suggest that Fra-1 orchestrates the inflammatory state of macrophages by inhibition of Arg1 expression and thereby impedes the resolution of inflammation.

Keywords: Arthritis; Cell Biology; Inflammation; Macrophages.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. GO enrichment analysis links Fra-1 in macrophages to cell proliferation, response to growth factors, and wounding.

Thioglycollate-elicited macrophages were isolated from Fra-1ΔMx and control mice (n = 2). (A) The deletion efficiency of Fra-1 was quantified by real time PCR (RT-PCR). Data are shown as mean of 2 samples with duplicates and error bars represent SEM. ***P < 0.01, Student’s t test. (B) Heatmap of differentially expressed genes ascertained from microarray analysis. (C–E) GO enrichment analysis of differentially expressed genes found in the microarray analysis (related to Supplemental Figure 2). Depicted are genes associated with the terms in the cluster.

Figure 2. Fra-1 modulates expression and activity of Arg1 and Nos2.

Thioglycollate-elicited macrophages or BMDMs were isolated from Fra-1ΔMx or control littermate mice. 1 × 106 Macrophages were stimulated with 50 ng/ml IFN-γ, 1 μg/ml LPS, 100 ng/ml IL-4, or 5 × 106 ACs for the indicated time points. (A) Fra-1 mRNA levels in WT thioglycollate-elicited macrophages were determined by RT-PCR. (B) Fra-1 protein levels in thioglycollate-elicited macrophages isolated from Fra-1ΔMx or control mice. Shown are representative data from 1 out of 3 experiments. (C) Fra-1 mRNA levels in WT BMDMs after stimulation. (D and E) Fra-1 mRNA levels in WT and mutant (D) thioglycollate-elicited macrophages or (E) BMDMs stimulated with LPS or AC. (F and G) Arg1 mRNA levels in WT and mutant (F) thioglycollate-elicited macrophages or (G) BMDMs stimulated with LPS or AC. (H and I) Nos2 mRNA levels in WT and mutant (H) thioglycollate-elicited macrophages or (I) BMDMs stimulated with LPS or AC. (J) Arginase activity was determined in cell lysates of thioglycollate-elicited macrophages (left) or BMDMs (right). (K) iNOS activity was determined by Griess assay in supernatants of WT and mutant thioglycollate-elicited macrophages (left) or BMDMs (right). Data are shown as mean values of 3 independent experiments, and error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, ANOVA.

Figure 3. Fra-1 transcriptionally regulates Arg1 at the promoter level.

Thioglycollate-elicited macrophages from Fra-1ΔMx or control littermate mice unstimulated or stimulated with LPS or AC were used for ChIP analysis. Chromatin was precipitated using an anti-mouse Fra-1 antibody or IgG isotype control; the obtained eluate was analyzed by sequencing or real-time PCR. (A and B) Peak of Fra-1 binding on the (A) Arg1 and (B) Nos2 promoters assessed by ChIP-Seq of LPS- or AC-stimulated WT macrophages. (C and D) AP-1 consensus sequences on the (C) Arg1 or the (D) Nos2 promoter were determined by the online tool TF search and are indicated by gray boxes. Additionally, the locations of the primers (arrows) are indicated. (E and F) Fra-1 ChIP analysis by real-time PCR for (E) Arg1 and (F) Nos2 promoters. The eluates arose from Fra-1ΔMx or control macrophages, and the Ct values are normalized to input. (G) Fra-1 ChIP analysis by real-time PCR for Arg1 and Nos2 promoters. The eluates arose from control macrophages stimulated for 1 hour with LPS or AC, and the Ct values normalized to the input were subsequently normalized to Fra-1 binding in unstimulated macrophages. (H) Jun protein ChIP analysis for Arg1 and Nos2 promoters. The eluates arose from control macrophages that were precipitated using anti-mouse JunB, JunD, cJun, or IgG isotype. Ct values were normalized to input. (I) AP-1 consensus sequences (Arg1-1/2/4/5/8 and Nos2-10) were cloned into a luciferase reporter construct and transfected into 293T cells; luciferase activity was determined. Mutated reporter constructs deleted for the respective binding sites were used as negative and renilla as internal control. The luciferase/renilla ratio was normalized to an empty pGL4.23 luciferase construct (n = 3). Data are shown as mean values of 3 independent experiments, and the error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA.

Figure 4. Ameliorated joint inflammation in Fra-1ΔMx arthritic mice.

K/BxN arthritis was induced in Fra-1 mutant mice and their respective control mice. Healthy or arthritic mice were analyzed 10 days after serum transfer. (A) Fra-1 mRNA levels in paws of healthy and arthritic control mice (n = 4). (B) Fra-1 mRNA levels in stromal cells (CD11b–), neutrophils (CD11b+Ly6G+), monocytes (CD11b+Ly6C+), and macrophages (CD11b+F4/80+) sorted from arthritic Fra-1ΔMx, Fra-1ΔLysM, and control littermate mice. (C) Arthritis scores of control, controlΔMx, and Fra-1ΔMx mice and quantification of AUC. (D) Quantification and representative images of paw volume ascertained from in vivo MRI analysis of healthy, arthritic Fra-1ΔMx and arthritic control mice. Graph points indicate individual mice. Data are shown as mean values, and the error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test (A and B) or ANOVA (C and D).

Figure 5. Fra-1 in myeloid cells exacerbates the development of K/BxN SIA inflammation in joints.

BM from Fra-1ΔMx or control mice was transferred to previously irradiated Fra-1ΔMx or WT mice (WT→WT, WT→Fra-1ΔMx, Fra-1ΔMx→WT, and Fra-1ΔMx→Fra-1ΔMx). Six weeks after BM reconstitution, K/BxN arthritis was induced in recipient mice. (A) Fra-1 expression in arthritic paws. (B) Arthritis score as well as quantification of the AUC. (C) Quantification and representative images of paw volume ascertained by MRI analysis of healthy control paws and arthritic paws from WT→WT and Fra-1ΔMx→WT mice. (D) Quantification of the inflammatory area, erosion area, and number of osteoclast and representative images ascertained from H&E (top) and TRAP (bottom) staining of arthritic paws. The arrows indicate cell infiltrated areas in H&E staining and osteoclasts in TRAP staining, respectively. Scale bars: 500 μm. (E) Representative μCT imaging analysis of arthritic ankles from WT→WT and Fra-1ΔMx→WT. The arrows indicate osteophyte formation. Graph points indicate individual mice. Data are also shown as mean values, and error bars represent SEM. *P < 0.05; ***P < 0.001, Student’s t test (A, C, D) or ANOVA (B).

Figure 6. Ameliorated joint inflammation in Fra-1ΔLysM arthritic mice.

K/BxN arthritis was induced in Fra-1ΔLysM and control littermate mice. (A) Arthritis score of control, controlΔLysM, and Fra-1ΔLysM mice and quantification of AUC. (B) Quantification of soft tissue volume and representative images ascertained by MRI analysis of healthy control and arthritic control and Fra-1ΔLysM mice. (C) Quantification of the inflammatory area, erosion area, and number of osteoclasts and representative images ascertained from H&E (top) and TRAP (bottom) staining of arthritic paws. The arrows indicate cell infiltrated areas in H&E staining and osteoclasts in TRAP staining, respectively. Scale bars: 500 μm. Graph points indicate individual mice. Data are shown as mean values, and error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test (B and C) or ANOVA (A).

Figure 7. Increased Arg1 in Fra-1–deleted macrophages from arthritic joints.

K/BxN arthritis was induced in Fra-1 mutant mice and their respective control mice. Healthy or arthritic mice were analyzed 10 days after serum transfer. (A) Arg1 mRNA levels were determined in paws of healthy, arthritic control and Fra-1 mutant mice (Fra-1ΔMx, Fra-1ΔMx→WT, and Fra-1ΔLysM). (B) The concentrations of ʟ-arg and ʟ-ornithine in paw lysates of Fra-1ΔMx and control littermate mice were analyzed by HPLC. (C) Arg-1 mRNA levels in stromal cells (CD11b–), neutrophils (CD11b+Ly6G+), monocytes (CD11b+Ly6C+), and macrophages (CD11b+F4/80+) sorted from arthritic Fra-1ΔMx, Fra-1ΔLysM, and control littermate mice. (D) Intracellular Arg1 protein levels observed as MFI in neutrophils, monocytes, and macrophages isolated from healthy control or arthritic joints of WT, Fra-1ΔMx, and Fra-1ΔLysM mice. Shown are ΔMFI as compared with unstained and normalized to healthy controls of each respective cell type. A representative histogram of MFI is shown for macrophages from healthy control mice, arthritic control mice, and arthritic Fra-1ΔMx mice. Graph points indicate individual mice. Data are shown as mean values, and error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA.

Figure 8. Arginase inhibition by NOHA restores arthritis in Fra-1ΔMx mice.

Arthritis was induced by K/BxN serum-transfer in Fra-1ΔMx or control mice. Mice were i.p. injected with NOHA (100 mg/kg body weight, Fra-1ΔMx or control) or PBS (Fra-1ΔMx or control) daily from the day of arthritis induction, and mice were analyzed 10 days after serum transfer. (A) Arg1 mRNA levels were quantified in whole paw. (B) Arg1 enzyme activity in whole-paw lysates was quantified by arginase activity assay. (C) Arthritis scores and AUC. (D) Quantification of the inflammatory area, erosion area, and number of osteoclasts from the histological analysis of H&E (top) and TRAP (bottom) staining and its representative images. The arrows indicate cell infiltrated areas in H&E staining and osteoclasts in TRAP staining, respectively. Scale bars: 500 μm. (E) Representative images of μCT imaging analysis (n = 3). The arrows indicate osteophyte formation. Graph points indicate individual mice. Data are shown as mean values, and error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA.

Figure 9. Therapeutic l-arg supplementation resolves arthritis.

Arthritis was induced by K/BxN serum transfer to WT mice, supplemented with 40 g/l ʟ-arg in the drinking water, either simultaneously with the K/BxN serum transfer (d0) or therapeutically at d4 after serum transfer. Mice were analyzed at day 10 after serum transfer. (A) Arg1 activity in total paw lysates. (B) Arthritis score and its quantification of AUC. (C) Quantification of the inflammatory area, erosion area, and number of osteoclasts from the histological analysis of H&E (top) and TRAP (bottom) staining and its representative images. Scale bars: 500 μm. The arrows indicate cell infiltrated areas in H&E staining and osteoclasts in TRAP staining, respectively. Graph points indicate individual mice. Data are shown as mean values, and error bars represent SEM. *P < 0.05; **P < 0.01; ***P < 0.001, ANOVA.

Figure 10. Fra-1 and Arg1 expression in the joints of RA patients.

(A) Fra-1 mRNA expression in peripheral blood cells of patients with RA at different levels of inflammatory disease activity measured by DAS28. DAS28 of less than 2.6 indicated low/no disease activity (n = 11), more than 2.7 and less than 5.1 indicated moderate disease activity (n = 11), and more than 5.2 indicated high disease activity (n = 12). (B) Correlation between mRNA expression of Fra-1 and Arg1 (left) or arginase activity (right) in peripheral blood cells or serum, respectively. Rel exp, relative expression. (C) Immunofluorescence image for CD68 (magenta), Fra-1 (green), Arg1 (red), and DAPI (blue) in joint sections from RA patients in remission (DAS28 = 1.81) and moderate/high disease activity (DAS28 = 4.01). Scale bars: 100 μm. Arrowheads point to CD68+ macrophages. White inset boxes are 3 times magnified from the original magnification. (D) CD68+ cells were quantified for their Fra-1/Arg1 ratio by the mean gray value in the synovial tissue of RA patients (n = 14) in remission (DAS28 < 2.6, n = 9) or moderate/high disease activity (DAS28>3.2, n = 5). Each point represents the ratio of the mean gray value (Fra-1/Arg1) per macrophage. (E) Schematic of Fra-1 actions in macrophages: Fra-1 blocks antiinflammatory responses in macrophages by the inhibition of the Arg1 pathways. *P < 0.05; **P < 0.01.